The goal of this competitive renewal is to identify the genetic basis in humans for cleft lip with or without cleft palate (CL/P), which is a common and genetically complex birth defect. We have accomplished all 3 of the specific aims of the initial grant, including: 1) Recruitment of more than 130 extended families and 500 affected child/parent trios. 2) Genetic analysis of candidate genes, which revealed positive results in both Colombian and Ohio families. These loci will be used as covariates in gene-gene interaction tests. 3) Completion of a genome wide linkage scan which identified additional positive loci, including a previously unidentified major locus at 9q22-33. A genome scan, accomplished by the successful application to the Center for Inherited Disease Research (CIDR: Lidral PI) in collaboration with Drs. Arcos-Burgos, Marazita and Murray to evaluate over 600 CL/P families from 7 different populations, revealed a highly significant result to 9q22-33 (combined LOD=6.6) in 4 of these populations with the Colombian families yielding the most significant results. The specific focus of the renewal is to positionally clone the 9q CL/P locus which has an 18cM 2-LOD interval. The initial fine-mapping will be accomplished by genotyping an Illumina panel of 1536 SNPs on the same 600 families to narrow the region by linkage and to screen for association. The genotyping will be performed by CIDR as part of a successful pilot project application (Lidral PI). For greater power we have established collaborations with another center in Colombia that is a unique admixed population of Native American Indians, Spaniards and Africans. This admixture likely results in a powerful pattern of linkage disequilibrium that permits association studies at a much lower marker density. We propose to explore whether this exists in our study population and if so apply emerging admixture methods. All total, we will be able to recruit a total of more than 300 extended and 2000 trio families that will be the basis for replication and for future studies to fine-map the other positive loci and identify gene-gene interactions. To aid in prioritizing which genes should be sequenced, we propose to determine in a mouse CL/P model the embryonic facial expression of uncharacterized genes within the critical region. Through the use of complimentary mouse and human genetics this project will identify genes and pathways essential for normal facial development, such that preventive strategies for CL/P can be developed.